Monocyte-macrophage colony-stimulating factor is produced by a variety of cells, including macrophages, endothelial cells and fibroblasts (see, Ralph et al., "The Molecular and Biological Properties of the Human and Murine Members of the CSF-1 Family" in Molecular Basis of Lymphokine Action, Humana Press, Inc., (1987), which is incorporated herein by reference). M-CSF is composed of two "monomer" polypeptides, which form a biologically active dimeric M-CSF protein (hereinafter referred to as "M-CSF dimer"). M-CSF belongs to a group of biological agonists that promote the production of blood cells. Specifically, it acts as a growth and differentiation factor for bone marrow progenitor cells of the mononuclear phagocyte lineage. Further, M-CSF stimulates the proliferation and function of mature macrophages via specific receptors on responding cells. In clinical trials M-CSF has shown promise as a pharmaceutical agent in the correction of blood cell deficiencies arising as a side-effect of chemotherapy or radiation therapy for cancer and may be beneficial in treating fungal infections associated with bone marrow transplants. M-CSF may also play significant biological roles in pregnancy, uveitis, and atherosclerosis. Development of M-CSF agonists or antagonists may prove to be of value in modifying the biological events involved in these conditions.
M-CSF exists in at least three mature forms: short (M-CSF.alpha.), intermediate (M-CSF.gamma.), and long (M-CSF.beta.). Mature M-CSF is defined as including polypeptide sequences contained within secreted M-CSF following amino terminus processing to remove leader sequences and carboxyl terminus processing to remove domains including a putative transmembrane region. The variations in the three mature forms are due to alternative mRNA splicing (see, Cerretti et al. Molecular Immunology, 25:761 (1988)). The three forms of M-CSF are translated from different mRNA precursors, which encode polypeptide monomers of 256 to 554 amino acids, having a 32 amino acid signal sequence at the amino terminal and a putative transmembrane region of approximately 23 amino acids near the carboxyl terminal. The precursor peptides are subsequently processed by amino terminal and carboxyl terminal proteolytic cleavages to release mature M-CSF. Residues 1-149 of all three mature forms of M-CSF are identical and are believed to contain sequences essential for biological activity of M-CSF. In vivo M-CSF monomers are dimerized via disulfide-linkage and are glycosylated. Some, if not all, forms of M-CSF can be recovered in membrane-associated form. Such membrane-bound M-CSF may be cleaved to release secreted M-CSF. Membrane associated M-CSF is believed to have biological activity similar to M-CSF, but may have other activities including cell-cell association or activation.
Polypeptides, including the M-CSFs, have a three-dimensional structure determined by the primary amino acid sequence and the environment surrounding the polypeptide. This three-dimensional structure establishes the polypeptide's activity, stability, binding affinity, binding specificity, and other biochemical attributes. Thus, a knowledge of a protein's three-dimensional structure can provide much guidance in designing agents that mimic, inhibit, or improve its biological activity in soluble or membrane bound forms.
The three-dimensional structure of a polypeptide may be determined in a number of ways. Many of the most precise methods employ X-ray crystallography (for a general review, see, Van Holde, Physical Biochemistry, Prentice-Hall, N. J. pp. 221-239, (1971), which is incorporated herein by reference). This technique relies on the ability of crystalline lattices to diffract X-rays or other forms of radiation. Diffraction experiments suitable for determining the three-dimensional structure of macromolecules typically require high-quality crystals. Unfortunately, such crystals have been unavailable for M-CSF as well as many other proteins of interest. Thus, high-quality, diffracting crystals of M-CSF would assist the determination of its three-dimensional structure.
Various methods for preparing crystalline proteins and polypeptides are known in the art (see, for example, McPherson, et al. "Preparation and Analysis of Protein Crystals", A. McPherson, Robert E. Krieger Publishing Company, Malabar, Fla. (1989); Weber, Advances in Protein Chemistry 41:1-36 (1991); U.S. Pat. No. 4,672,108; and U.S. Pat. No. 4,833,233; all of which are incorporated herein by reference for all purposes). Although there are multiple approaches to crystallizing polypeptides, no single set of conditions provides a reasonable expectation of success, especially when the crystals must be suitable for X-ray diffraction studies. Thus, in spite of significant research, many proteins remain uncrystallized.
In addition to providing structural information, crystalline polypeptides provide other advantages. For example, the crystallization process itself further purifies the polypeptide, and satisfies one of the classical criteria for homogeneity. In fact, crystallization frequently provides unparalleled purification quality, removing impurities that are not removed by other purification methods such as HPLC, dialysis, conventional column chromatography, etc. Moreover, crystalline polypeptides are often stable at ambient temperatures and free of protease contamination and other degradation associated with solution storage. Crystalline polypeptides may also be useful as pharmaceutical preparations. Finally, crystallization techniques in general are largely free of problems such as denaturation associated with other stabilization methods (e.g. lyophilization). Thus, there exists a significant need for preparing M-CSF compositions in crystalline form and determining their three-dimensional structure. The present invention fulfills this and other needs. Once crystallization has been accomplished, crystallographic data provides useful structural information which may assist the design of peptides that may serve as agonists or antagonists. In addition, the crystal structure provides information useful to map, the receptor binding domain which could then be mimicked by a small non-peptide molecule which may serve as an antagonist or agonist.